MEKK3稳定表达细胞株的建立及其对A549细胞增殖的影响

目的 构建人MEKK3基因编码区序列（cDNA）的真核表达载体、建立其稳定表达细胞株并观察其对肺腺癌细胞增殖的影响.方法 从A549细胞中提取总RNA,应用RT-PCR扩增MEKK3 cDNA的全长序列后克隆入pcDNA3.1/hygro（＋）质粒中,构建成MEKK3基因的真核表达载体,然后转染入人肺腺癌A549细胞中,潮霉素筛选稳定转染克隆,通过MTT实验,研究转染MEKK3基因前后细胞增殖的变化.结果 重组载体经酶切鉴定和测序证实目的 基因正确无误,Western印迹检测结果显示MEKK3基因在A549细胞中具有良好的表达;荧光实时定量PCR结果表明MEKK3基因在其稳定转染的A549细胞克隆中表达上调,与空载体稳定转染及未转染细胞比较,差异具有统计学意义（P＜0.05）;MTT结果显示MEKK3表达上调的稳定克隆组,A549细胞的增殖活性显著增强（A570=0.876 1±0.074 5）,明显高于空载体稳定转染组（A570=0.582 8±0.070 3）及未转染亲代细胞组（A570=0.584 9±0.035 2）,差异具有统计学意义（P＜0.01）,而后两者之间差异无统计学意义（P＞0.05）.结论 MEKK3表达上调可导致肺腺癌细胞的增殖增强.

Establishment of a Cell Line Stably Expressing MEKK3 and Effect of MEKK3 on Proliferation of A549 Cells

Objective To construct eukaryotic expression vector pcDNA3.1/hygro （ ＋ ） - MEKK3, establish cell clones stably expressing MEKK3 and study the effect of MEKK3 on proliferation of lung adenocarcinoma cells. Methods Total RNA was extracted from A549 lung cancer cells. MEKK3 eDNA was amplified by RT-PCR, and inserted into pcDNA3.1/hygro （ ＋ ） vector to construct pcDNA3, l/hygro （ ＋ ） -MEKK3. The recombinant plasmid was transfected into A549 cells by lipofectamine. The stable transfectants were obtained with hygromycin B. Western blot and real-time PCR were used to detect the expression of MEKK3 protein and mRNA. MTT assay was used to evaluate the effects of MEKK3 on proliferation of A549 cells. Results The recombinant expres- sion vector pcDNA3.1/ hygro （ ＋ ） -MEKK3 was successfully constructed. The expression of MEKK3 protein was significantly higher in A549 cells transfected with the MEKK3 expression vector than in those transfected with empty vector or untransfected A549 ceils. In addition, the expression of MEKK3 mRNA was significantly higher in stable transfectants with the MEKK3 vector than in those with empty vector or in untransfected cells （ P 〈 0.05 ）, and there was no significant difference between the latter two groups （P 〉 0. 05） . By MTT assay, we found that the proliferation activity of A549 cells in stably transfected group with the MEKK3 expression vector （A570 = 0. 8761 ± 0. 0745） was significantly stronger than in those with empty vector （A570 =0. 5828 ±0. 0703） or in untransfected cells （A570 -0. 5849±0. 0352） （P 〈0.01）, and there was no significant difference between the latter two groups （P 〉 0.05） . Conclusion MEKK3 can increase proliferation of lung adenocareinoma eells.